Integrin-targeting vectors having transfection activity

ABSTRACT

A complex that comprises (i) a nucleic acid, (ii) an integrin-binding component, for example, an integrin-binding peptide, (iii) a polycationic nucleic acid-binding component, for example, oligolysine, and (iv) a lipid component, for example, a cationic liposome, has transfection activity.

[0001] The present invention relates to an improved integrin-targetingvector that has enhanced transfection activity.

[0002] Gene therapy and gene vaccination are techniques that offerinteresting possibilities for the treatment and/or prophylaxis of avariety of conditions, as does anti-sense therapy. Such techniquesrequire the introduction of a DNA of interest into target cells. Theability to transfer sufficient DNA to specific target cells remains oneof the main limitations to the development of gene therapy, anti-sensetherapy and gene vaccination. Both viral and non-viral DNA deliverysystems have been proposed. In some cases RNA is used instead of DNA.

[0003] Receptor-mediated gene delivery is a non-viral method of genetransfer that exploits the physiological cellular process,receptor-mediated endocytosis to internalise DNA. Receptor-mediatednon-viral vectors have several advantages over viral vectors. Inparticular, they lack pathogenicity; they allow targeted gene deliveryto specific cell types and they are not restricted in the size ofnucleic acid molecules that can be packaged. Gene expression is achievedonly if the nucleic acid component of the complex is released intactfrom the endosome to the cytoplasm and then crosses the nuclear membraneto access the nuclear transcription machinery. However, transfectionefficiency is generally poor relative to viral vectors owing toendosomal degradation of the nucleic acid component, failure of thenucleic acid to enter the nucleus and the exclusion of aggregates largerthan about 150 nm from clathrin coated vesicles.

[0004] Integrins are a super-family of heterodimeric membrane proteinsconsisting of several different α and β subunits. They are important forattachment of cells to the extracellular matrix, cell-cell interactionsand signal transduction. Integrin-mediated cell entry is exploited forcell attachment and entry by a number of intracellular pathogensincluding Typanosoma cruzi (Fernandez et al., 1993), adenovirus (Wickhamet al., 1993), echovirus (Bergelson et al., 1992) and foot-and-mouthdisease virus (Logan et al., 1993) as well as the enteropathogen Y.pseudotuberculosis (Isberg, 1991). Egg-sperm fusion is also integrinmediated. Intensive study of the invasin-integrin mediatedinternalisation process of Yersinia pseudotuberculosis demonstratedthat, for efficient cell entry, integrin-binding ligands should have ahigh binding affinity and a non-polar distribution (Isberg, 1991).Integrin-mediated internalisation proceeds by a phagocytic-like processallowing the internalisation of bacterial cells one to two micrometersin diameter (Isberg, 1991). Targeting of non-viral vectors to integrins,therefore, has the potential to transfect cells in a process that mimicsinfection of cells by pathogens and avoids the size limitation imposedby clathrin-coated vesicles in receptor-mediated endocytosis.

[0005] A further advantage of integrin-mediated vectors is that a largenumber of peptide ligands for integrin receptors have been described,including sequences derived from natural protein ligands [Verfaille,1994 #635; Wang, 1995 #645; Staatz, 1991 #539; Pierschbacher, 1984 #314;Massia, 1992 #86, Clements et al. 1994 & Lu et al, 1993] or selectedfrom phage display libraries (Koivunen et al. 1995; 1993; 1994; O'Neilet al. 1992; Healy et al 1995; Pasqualani et al. 1995).

[0006] The conserved amino acid sequence arginine-glycine-aspartic acid(RGD) is an evolutionarily conserved feature of many, but not all,natural integrin-binding ligands such as extracellular matrix proteinsand viral capsids. Peptides, particularly those containing cyclic-RGDdomains can also bind integrins. Peptides containing cyclic-RGD domainsare particularly suitable ligands for vectors since they bind tointegrins with higher affinities than linear peptides (Koivunen et al.1995). Hart et al. have demonstrated previously that multiple copies ofa cyclic RGD peptide displayed in the major coat protein subunit of fdfilamentous phage particles, approximately 900 nm in length, areinternalised efficiently by cells in tissue culture in anintegrin-mediated manner (Hart et al., 1994). The phage particles wereprobably internalised by a phagocytic-like process as their size wouldexclude them from endocytosed vesicles (Hart et al., 1994).

[0007] The cyclic RGD-containing peptide GGCRGDMFGCGG[K]₁₆[SEQ.ID.NO.:1] was synthesised with a sixteen-lysine tail for complexformation with plasmid DNA (Hart et al., 1995). Significant levels ofintegrin-mediated gene expression were achieved in epithelial cell lineswith the vector GGCRGDMFGCG[K]₁₆ [SEQ.ID.NO.:2] (Hart et al., 1995) andthe vectors GGCRGDMFGC[K]₁₆ [SEQ.ID.NO.:3] (WO96/15811). A similarpeptide [K]₁₆GACRGDMFGCA [SEQ.ID.NO.:4], which has the sixteen-lysinedomain at the N-terminus and which is easier to synthesise than theprototype peptide (WO96/15811 and Hart et al., 1997) generated bettertransfection levels. Integrin mediated gene expression was generallyachieved at levels of about 1 to 10%. The presence of chloroquine in thetransfection medium gave some enhancement of transfection in some butnot all cell lines tested.

[0008] The present invention is based on the surprising observation thatinclusion of a lipid component in the oligolysine/peptide/DNA complexincreases levels of transfection of DNA from about 1 to 10% to about 50to almost 100%. Not only is the level of transfection increaseddramatically but, contrary to previous experience, the increase isobserved in all cell lines tested, including endothelial, epithelial andtumour cell lines.

[0009] The present invention provides a complex that comprises

[0010] (i) a nucleic acid, especially a nucleic acid encoding a sequenceof interest,

[0011] (ii) an integrin-binding component,

[0012] (iii) a polycationic nucleic acid-binding component, and

[0013] (iv) a lipid component.

[0014] The complex is a transfection vector.

[0015] The nucleic acid may be obtained from natural sources, or may beproduced recombinantly or by chemical synthesis. It may be modified, forexample, to comprise a molecule having a specific function, for example,a nuclear targeting molecule. The nucleic acid may be DNA or RNA. DNAmay be single stranded or double stranded. The nucleic acid may besuitable for use in gene therapy, in gene vaccination or in anti-sensetherapy. The nucleic acid may be or may relate to a gene that is thetarget for particular gene therapy or may be a molecule that canfunction as a gene vaccine or as an anti-sense therapeutic agent. Thenucleic acid may be or correspond to a complete coding sequence or maybe part of a coding sequence.

[0016] Alternatively, the nucleic acid may encode a protein that iscommercially useful, for example industrially or scientifically useful,for example an enzyme; pharmaceutically useful, for example, a proteinthat can be used therapeutically or prophylactically as a medicament orvaccine; or diagnostically useful, for example, an antigen for use in anELISA. Host cells capable of producing commercially useful proteins aresometimes called “cell factories”.

[0017] Appropriate transcriptional and translational control elementsare generally provided. For gene therapy, the nucleic acid component isgenerally presented in the form of a nucleic acid insert in a plasmid orvector. In some cases, however, it is not necessary to incorporate thenucleic acid component in a vector in order to achieve expression. Forexample, gene vaccination and anti-sense therapy can be achieved using anaked nucleic acid.

[0018] The nucleic acid is generally DNA but RNA may be used in somecases, for example, in cancer vaccination. The nucleic acid component isreferred to below as the plasmid component or component “D”.

[0019] The integrin-binding component is any component that is capableof binding specifically to integrins found on the surface of cells. Theintegrin-binding component may be a naturally occurring integrin-bindingligand, for example, an extra-cellular matrix protein, a viral capsidprotein, the bacterial protein invasin, a snake venom disintegrinprotein, or an integrin-binding fragment of any such protein. Suchintegrin-binding proteins and fragments thereof may be obtained fromnatural sources or by recombinant techniques, but they are difficult tosynthesise and purify in large amounts, they require conjugationdirectly to DNA or RNA or to polycationic elements for DNA or RNAbinding, and are immunogenic in vivo.

[0020] It is preferable to use integrin-binding peptides, in particularbecause of their ease of synthesis, purification and storage, theirpotential for chemical modification, and their potentially lowimmunogenicity in vivo. Examples of integrin-binding peptides are givenin Verfaille, 1994 #635; Wang, 1995 #645; Staatz, 1991 #539;Pierschbacher, 1984 #314; Massia, 1992 #86, Clements et al. 1994 & Lu etal, 1993; and in Koivunen et al. 1995; 1993; 1994; O'Neil et al. 1992;Healy et al 1995; and Pasqualani et al. 1995.

[0021] As indicated above, peptides containing the conserved amino acidsequence arginine-glycine-aspartic acid (RGD) bind with high affinity tointegrins. Accordingly, peptides comprising the RGD sequence areparticularly useful. The affinity between integrin and peptide ligandsis influenced by the amino acid sequence flanking the RGD domain.Peptides having a cyclic region in which the conformational freedom ofthe RGD sequence is restricted generally have a higher affinity forintegrin receptors than do their linear counterparts. Such cyclicpeptides are particularly preferred. Cyclic peptides may be formed bythe provision of two cysteine residues in the peptide, thus enabling theformation of a disulphide bond. A cysteine residue may be separated fromthe RGD sequence by one or more residues, for example, up to sixresidues, or may be immediately adjacent to the RGD sequence, althoughpreferably both cysteines are not immediately adjacent to the ends ofthe RGD sequence.

[0022] An example of an amino acid sequence that will permit cyclisationby disulphide bond formation is CRGDMFGC [SEQ.ID.NO.:5]. A peptide thatconsists of or comprises the sequence CRGDMFGC may advantageously beused as an integrin-binding peptide according to the present invention.Examples of peptides that comprises the sequence CRGDMFGC and that areeffective integrin-binding ligands are the peptides GGCRGDMFGC[SEQ.ID.NO.:6], GGCRGDMFGCG [SEQ.ID.NO.:7], GGCRGDMFGCA [SEQ.ID.NO.:8]and GACRGDMFGCA [SEQ.ID.NO.:9].

[0023] The peptide GACDCRGDCFCA [SEQ.ID.NO.:10] has the potential toform two disulphide bonds for stabilising the RGD loop. That peptide andothers having the potential to form two RGD-stabilising disulphidebonds, may be particularly useful as integrin-binding ligands accordingto the present invention.

[0024] However, not all integrin-binding peptides contain the conservedRGD sequence. For example, the peptides GACRRETAWACA [SEQ.ID.NO.:11] andGACRRETAWACG [SEQ.ID.NO.:12] are integrin-specific peptides. Otherpeptides comprising the sequence CRRETTAWAC [SEQ.ID.NO.:13] may be used,as may other non-RGD peptides, particularly those that have thepotential for disulphide bond formation.

[0025] Peptide sequences may be designed on the basis of known ligands,for example, on the basis of integrin-binding domains ofnaturally-occurring integrin-binding ligands, or on the basis of knownpeptides that bind to integrins.

[0026] As stated above integrins are a family of heterodimeric proteinsfound on the surface of cells. They consist of several different α and βsubunits. Some integrins are found on may types of cells, others aremore specific, for example, α5 and αv integrins are widespread and arefound on a diverse range of cells. Integrin-binding ligands can vary intheir affinity for different integrins. For example, GACRGDMFGCA[SEQ.ID.NO.:9] (peptide 1) has affinity for α5 and αv integrins but isnon-specific (O'Neil et al. 1992, Hart et al. 1997). GACDCRGDCFCA[SEQ.ID.NO.:10] (peptide 5) has high affinity for integrin αv but is notαv-specific (Koivunen et al. 1995; Hart et al. 1997). GACRRETAWACG[SEQ.ID.NO.:11] (peptide 6) however, which does not contain theconserved RGD region, is α5β1-specific (Koivunen et al. 1995). Variousintegrin-binding peptides and their integrin specificity are set out inthe Table below: TABLE Peptide number and integrin specificity SequenceSEQ.ID.NO. Peptide 1 (αv,a5β1) GACRGDMFGCA SEQ.ID.NO.:9 Peptide 2(αv,α5β1) GACRGDMFGCGG SEQ.ID.RO.:12 Peptide 5 (αv) GACDCRGDCFCASEQ.ID.NO.:10 Peptide 6 (α5β1) GACRRETAWACG SEQ.ID.NO.:11 Peptide 7(α4β1) GAGPEILDVPST SEQ.ID.NO.:13 Peptide 8 (α4β1) GACQIDSPCASEQ.ID.NO.:14 Peptide 9 (α5β1) GACRRETAWACGKGACRRETAWACG SEQ.ID.NO.:15

[0027] It should be noted that the use of a lipid component according tothe present invention greatly enhances transfection for all peptides andall cell types tested, unlike other enhancement techniques that havebeen tried, for example, chloroquine, which enhance transfection to asmall extent in some but not all cell types tested.

[0028] The polycationic nucleic acid-binding component is any polycationthat is capable of binding to DNA or RNA. The polycation may have anynumber of cationic monomers provided the ability to bind to DNA or RNAis retained. For example, from 3 to 100 cationic monomers may bepresent, for example, from 10⁻ to 20, especially about 16. Anoligolysine is particularly preferred, for example, having from 10 to 20lysine residues, for example, from 15 to 17 residues, especially 16residues i.e. [K]₁₆.

[0029] The polycationic DNA or RNA-binding component may advantageouslybe linked or otherwise attached to the integrin-binding component. Acombined integrin-binding component/polycationic DNA or RNA-bindingcomponent may be referred to below as component “I”. For example, apolycationic DNA or RNA-binding component may be chemically bonded to anintegrin-binding component, for example, by a peptide bond in the caseof an oligolysine. The polycationic component may be linked at anyposition of the integrin-binding component. Preferred combinations ofintegrin-binding component and polycationic DNA or RNA-binding componentare an oligolysine, especially [K]₁₆, linked via a peptide bond to apeptide, for example, a peptide as described above.

[0030] The lipid component may be or or may form a cationic liposome.The lipid component may be or may comprise one or more lipids selectedfrom cationic lipids and lipids having membranae destabilising orfusogenic properties, especially a combination of a cationic lipid and alipid that has membrane destabilising properties.

[0031] A preferred lipid component (“L”) is or comprises the neutrallipid dioleyl phosphatidylethanolamine, referred to herein as “DOPE”.DOPE has membrane destabilising properties sometimes referred to as“fusogenic” properties (Farhood et al. 1995). Other lipids, for example,neutral lipids, having membrane destabilising properties, especiallymembrane destabilising properties like those of DOPE may be used insteadof or as well as DOPE.

[0032] Other phospholipids having at least one long chain alkyl group,for example, di(long alkyl chain)phospholipids may be used. Thephospholipid may comprise a phosphatidyl group, for example, aphosphatidylalkanolamine group, for example, a phosphatidylethanolaminegroup.

[0033] A further preferred lipid component is or comprises the cationiclipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride,referred to herein as “DOTMA”. DOTMA has cationic properties. Othercationic lipids may be used in addition to or as an alternative toDOTMA, in particular cationic lipids having similar properties to thoseof DOTMA. Such lipids are, for example, quaternary ammonium saltssubstituted by three short chain alkyl groups, and one long chain alkylgroup. The short chain alkyl groups may be the same or different, andmay be selected from methyl and ethyl groups. At least one and up tothree of the short chain alkyl group may be a methyl group. The longalkyl chain group may have a straight or branched chain, for example, adi(long chain alkyl)alkyl group.

[0034] Another preferred lipid component is or comprises the lipid2,3-dioleyloxy-N-[2-(spermidinecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoridoacetate,referred to herein as “DOSPA”. Analogous lipids may be used in additionto or as an alternative to DOSPA, in particular lipids having similarproperties to those of DOSPA. Such lipids have, for example, differentshort chain alkyl groups from those in DOSPA.

[0035] A preferred lipid component comprises DOPE and one or more otherlipid components, for example, as described above. Especially preferredis a lipid component that comprises a mixture of DOPE and DOTMA. Suchmixtures form cationic liposomes. An equimolar mixture of DOPE and DOTMAis found to be particularly effective. Such a mixture is knowngenerically as “lipofectin” and is available commercially under the name“Lipofectin”. The term “lipofectin” is used herein generically to denotean equimolar mixture of DOPE and DOTMA. Other mixtures of lipids thatare cationic liposomes having similar properties to lipofectin may beused. Lipofectin is particularly useful as it is effective in all celltypes tested.

[0036] A further preferred lipid component comprises a mixture of DOPEand DOSPA. Such mixtures also form cationic liposomes. A mixture of DOPEand DOSPA in a ratio by weight 3:1 DOSPA:DOPE is particularly effective.Such a mixture, in membrane filtered water, is available commerciallyunder the name “Lipofectamine”. Mixtures comprising DOPE, DOTMA andDOSPA may be used, for example, mixtures of lipofectin andlipofectamine.

[0037] Other cationic lipids are available commercially, for example,DOTAP (Boehringer-Mannheim) and lipids in the Tfx range (Promega). DOTAPis N-[1-(2,3-diolyloxy)propyl]-N,N,N-trimethylammonium methylsulphate.The Tfx reagents are mixtures of a synthetic cationic lipid[N,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediammoniumiodide and DOPE. All the reagents contain the same amount of thecationic lipid component but contain different molar amounts of thefusogneic lipid, DOPE.

[0038] However, lipofectin and lipofectamine appear to be markedly moreeffective as the lipid component in LID complexes of the presentinvention than are DOTPA and Tfx agents.

[0039] The effectiveness of a putative integrin-binding component,polycationic DNA or RNA-binding component, or of lipid component may bedetermined readily using the methods described herein.

[0040] The efficiency of transfection using a complex of the inventionis influenced by the ratio lipid component:integrin-bindingcomponent:DNA or RNA. For any chosen combination of components for anyparticular type of cell to be transfected, the optimal ratios can bedetermined simply by admixing the components in different ratios andmeasuring the transfection rate for that cell type, for example, asdescribed herein.

[0041] For example, a combination consisting of a pGL2 plasmid, which isa plasmid encoding luciferase (a reporter gene) under an SV40 promoteras DNA component (D), [K]₁₆GACRGDMFGCA (SEQ.ID.NO.:17] ([K]₁₆-peptide 1)as a combined integrin-binding component/polycationic DNA bindingcomponent (I), and lipofectin (DOPE:DOTMA 1:1 molar ratio) as the lipidcomponent (L) was tested to find the optimal ratio of components.Complexes formed with 1 μg of lipofectin (L) and 4 μg of [K]₁₆-peptide(I) per 1 μg of plasmid (D) were 100-fold more active than complexeslacking lipofectin. Addition of larger amounts of lipofectin reducedtransfection activity in a lipofectin dose-dependent manner.

[0042] An optimal transfection ratio of 0.75 μg of lipofectin (L) per 4μg of the [K]₁₆-peptide integrin-binding component/-polycationic DNA orRNA-binding component (I) per 1 μg plasmid DNA or RNA (nucleic acidcomponent, D) was found for three different cell lines namely melanomacell, endothelial cells and epithelial cells. That ratio wassubsequently found to be effective for other different cell lines andfor other oligolysine-peptides. A ratio L:I:D of 0.75:4:1 by weightcorresponds to a molar ratio of 0.5 nmol lipofectin: 1.25 nmol[K]₁₆-peptide 6: 0.25 pmol plasmid pGL2-control. A ratio L:I:D of0.75:4:1 by weight, or the corresponding molar ratio are preferred whenlipofectin is used as the lipid component.

[0043] For a combination of components in which lipofectin is replacedby lipofectamine (DOPE/DOSPA), the optimal ratio was found to be 12 μglipofectamine: 4 μg [K]₁₆-peptide 6: 1 μg plasmid DNA or RNA. A ratio ofL:I:D of 12:4:1 by weight, or the corresponding molar ratio, isappropriate for lipofectamine-containing complexes. Optimal ratios forother systems may be determined analogously.

[0044] Lipofectin and lipofectamine appear to be particularly effectivein enhancing transfection. Lipofectin has the advantage that only verysmall amounts are required. Any side effects that may occur aretherefore minimised. As indicated above, the optimal weight ratio ofcomponents L:I:D when using lipofectamine is 12:4:1. With lipofectin theoptimal ratio is only 0.75:4:1.

[0045] The present invention provides a process for the production of atransfection complex of the present invention, which comprises admixingcomponents (i), (ii), (iii) and (iv).

[0046] Although the components may be admixed in any order, it isgenerally preferable that the lipid component is not added last. In thecase where there is a combined integrin-binding component/polycationicDNA or RNA-binding component it is generally preferable to combine thecomponents in the following order: lipid component; combinedintegrin-binding/polycationic DNA or RNA-binding component; DNA or RNAcomponent, for example, in the order: lipofectin, oligolysine-peptidecomponent, DNA or RNA component.

[0047] The present invention also provides a mixture comprising anintegrin-binding component, a polycationic nucleic acid-bindingcomponent, and a lipid component.

[0048] Such a mixture may be used to produce a nucleic acid-containingtransfection complex of the invention by the incorporation of a nucleicacid with the mixture, for example, by admixture. Alternatively, themixture of the invention may be used for the production of a complexwhich comprises, instead of the nucleic acid component, any othercomponent that is capable of binding to the polycationic nucleic acidbinding component, for example, a protein.

[0049] The present invention further provides a process for theproduction of a complex of the present invention, which comprisesadmixing a nucleic acid with a mixture of the invention.

[0050] The individual components of a mixture of the invention are eachas described above in relation to the complex of the invention. Thepreferred components, preferred combinations of components, preferredratios of components and preferred order of mixing, both with regard tothe mixture and to the production of a complex, are as described abovein relation to the complex of the invention.

[0051] A mixture of the present invention preferably comprises anequimolar mixture of DOPE and DOTMA (lipofectin) as the lipid componentand an oligolysine-peptide especially a [K]₁₆-peptide as a combinedintegrin-binding/nucleic acid-binding component. The preferred molarratio lipofectine:oligolysine-peptide is 0.75:4.

[0052] The present invention provides a method of transfecting a cellwith a nucleic acid, which comprises contacting the cell in vitro or invivo with a complex of the present invention.

[0053] The present invention also provides a process for expressing anucleic acid in a host cell, which comprises bringing the cell intocontact with a complex of the present invention. The host cell is thencultured under conditions that enable the cell to express the nucleicacid.

[0054] The present invention further provides a process for theproduction of a protein, which comprises contacting a host cell in vitroor in vivo with a complex of the present invention, allowing the cell toexpress the protein, and obtaining the protein. The host cell may betransfected in vitro with a nucleic acid by means of a complex of thepresent invention and cultured, the protein being obtained either fromthe host cell or from the culture medium.

[0055] The present invention further provides a cell transfected with acomplex of the present invention, and also the progeny of such a cell.

[0056] The present invention also provides a pharmaceutical compositionwhich comprises a complex of the present invention in admixture orconjunction with a pharmaceutically suitable carrier. The compositionmay be a vaccine.

[0057] The present invention also provides a method for the treatment orprophylaxis of a condition caused in a human or in a non-human animal bya defect and/or a deficiency in a gene, which comprises administering acomplex of the present invention to the human or to the non-humananimal.

[0058] The present invention also provides a method for therapeutic orprophylactic immunisation of a human or of a non-human animal, whichcomprises administering a complex of the present invention to the humanor to the non-human animal.

[0059] The present invention also provides a method of anti-sensetherapy of a human or of a non-human animal, wherein a complex of thepresent invention comprising anti-sense DNA is administered to the humanor to the non-human animal.

[0060] The present invention further provides a complex of the presentinvention for use as a medicament and/or vaccine, for example for theprophylaxis of a condition caused in a human or in a non-human animal bya defect and/or a deficiency in a gene, for therapeutic or prophylacticimmunisation of a human or of a non-human animal, or for anti-sensetherapy of a human or of a non-human animal.

[0061] The present invention also provides the use of a complex of thepresent invention for the manufacture of a medicament for theprophylaxis of a condition caused in a human or in a non-human animal bya defect and/or a deficiency in a gene, for therapeutic or prophylacticimmunisation of a human or of a non-human animal, or for anti-sensetherapy of a human or of a non-human animal.

[0062] A non-human animal is, for example, a mammal, bird or fish, andis particularly a commercially reared animal.

[0063] The DNA or RNA in the complex of the invention is appropriate forthe intended gene therapy, gene vaccination, or anti-sense therapy. TheDNA or RNA and hence the complex is administered in an amount effectivefor the intended purpose.

[0064] In a further embodiment, the present invention provides a kitsuitable for preparing a mixture of the present invention. Such a kitcomprises the following: (i) an integrin-binding component; (ii) apolycationic nucleic acid-binding component, and (iii) a lipidcomponent.

[0065] A kit suitable for producing a complex of the present inventionmay comprise components (i) to (iii) above and (iv) either a nucleicacid or a plasmid or vector suitable for the expression of a nucleicacid, the plasmid or vector being either empty or comprising the nucleicacid.

[0066] The components of a kit are, for example, as described above inrelation to a complex or a mixture of the present invention. Preferredcomponents are as described above.

[0067] A kit generally comprises instructions for the production of acomplex or a mixture of the present invention. The instructionspreferably indicate the preferred ratios of the components and thepreferred order of admixing the components, for example, as describedabove. A kit may be used for producing a complex suitable for genetherapy, gene vaccination or anti-sense therapy. Alternatively, it maybe used for producing a complex suitable for transfecting a host cellwith a nucleic acid encoding a commercially useful protein i.e. toproduce a so-called “cell factory”.

[0068] The kit of the present invention enables the user to producequickly and easily a highly efficient transfection complex of thepresent invention using any DNA or RNA of choice.

[0069] A kit of the invention may comprises the following components:

[0070] (a) an integrin-binding component, (b) a polycationic nucleicacid-binding component, (c) a lipid component and (d) a nucleic acid.

[0071] Such a kit is suitable for the production of a complex for use,for example, in gene vaccination or anti-sense therapy.

[0072] In a kit of the invention the components including the preferredcomponents are, for example, as described above in relation to a complexof the present invention.

[0073] The present invention also provides a lipid component asdescribed above for use in increasing the efficiency of transfection ofa cell with a nucleic acid, either DNA or RNA, the lipid component beingused in combination with an integrin-binding component and apolycationic nucleic acid-binding component.

[0074] The present invention also provides the use of a lipid componentas described above for the manufacture of a medicament comprising

[0075] (i) a nucleic acid, especially a nucleic acid encoding a sequenceof interest,

[0076] (ii) an integrin-binding component,

[0077] (iii) a polycationic nucleic acid-binding component and

[0078] (iv) the lipid component.

[0079] The medicament may be for gene therapy, gene vaccination, oranti-sense therapy.

[0080] The present invention also provides a transfection complex thatcomprises

[0081] (i) a nucleic acid, especially a nucleic acid encoding a sequenceof interest,

[0082] (ii) an integrin-binding component, and

[0083] (iii) a polycationic a nucleic acid-binding component,characterised in that a lipid component, for example as described above,is an additional component of the complex.

[0084] The present invention also provides a method for increasing theefficiency of a transfection vector that comprises

[0085] (i) a nucleic acid, especially a nucleic acid encoding a sequenceof interest,

[0086] (ii) an integrin-binding component, and

[0087] (iii) a polycationic a nucleic acid-binding component,characterised in that a lipid component, for example as described above,is incorporated as an additional component of the complex.

[0088] In each case, the various components are as described above. Thelipid component is, for example, a mixture of DOPE and DOSPA or,especially, a mixture of DOPE and DOTMA, in particular an equimolarmixture of DOPE and DOTMA (lipofectin).

[0089] Targets for gene therapy are well known and include monogenicdisorders, for example, cystic fibrosis, various cancers, andinfections, for example, viral infections, for example, with HIV. Forexample, transfection with the p53 gene offers great potential forcancer treatment. Targets for gene vaccination are also well known, andinclude vaccination against pathogens for which vaccines derived fromnatural sources are too dangerous for human use and recombinant vaccinesare not always effective, for example, hepatitis B virus, HIV, HCV andherpes simplex virus. Targets for anti-sense therapy are also known.Further targets for gene therapy and anti-sense therapy are beingproposed as knowledge of the genetic basis of disease increases, as arefurther targets for gene vaccination.

[0090] Transfection complexes of the present invention have beendemonstrated to transfect various different cell types, includingendothelial and epithelial cells, and tumour cells. Transfection of allcell types tested including cell types that are particularly reistant totransfection with most plasmid transfection vectors, for example,neuroblastoma cells, primary smooth muscle cells and cardiac myocytes,and haematopoieic cells has been achieved with high efficiency usingtransfection complexes of the present invention. This enables effectivegene therapy, gene vaccination and anti-sense therapy without theprevious restrictions as to cell type. For example, transfection withthe p53 gene for cancer therapy has great potential but is currentlylimited by the range of cell types in which effective transfection canbe achieved.

[0091] The effective tranfection of neuroblastoma cells demonstratesthat the complexes of the invention may be used as vaccines or fortherapy of neuroblastoma, an important childhood malignancy. Theeffective transfection of primary smooth muscle cells and cardiacmyocytes, which are particularly resistant to plasmid-mediatedtransfection, demonstrates that diseases and other pathologicalconditions affecting muscles and the cardiovascular system can now betreated by gene therapy. One such condition is restenosis. After balloonangioplasty plaques reform in 30-50% of cases. A gene that preventsproliferation of cells in blood vessel walls may be introduced using acomplex of the present invention to reduce restenosis.

[0092] Haematopoietic cells are another cell type that is particularlyresistant to plasmid-mediated transfection. The effectiveness oftranfection using a complex of the present invention, which can exceed60%, now enables gene therapy, gene vaccination and anti-sense therapyof diseases involving haematopoietic cells, including leukaemia and bonemarrow stem cell disorders. For example, transfection of a cytokine genemay be used for adjuvant immunotherapy.

[0093] Complexes of the invention have been demonstrated to be effectivevectors for intracellular transport and delivery of anti-senseoligonucleotides, which enables antiviral and cancer therapy.

[0094] Furthermore, complexes of the invention have been demonstrated tobe effective for intracellular transport of very large DNA molecules,for example, DNA larger than 125 kb, which is particularly difficultusing conventional vectors. This enables the introduction of artificialchromosomes into cells.

[0095] Transfection at high levels has been demonstrated in vivo,confirming the utility of the complexes of the invention for genetherapy, antisense therapy and gene vaccination. Transfection of theairways, for example, the bronchial epithelium demonstrates utility forgene therapy of, for example, cystic fibrosis and asthma. Transfectionof corneal endothelium demonstrates utility for treatment of eye diseaseaffecting the cornea or corneal organ transplants, for example inglaucoma.

[0096] The high levels of transfection make the complex of the inventionparticularly suitable for the production of host cells capable ofproducing a desired protein, so-called “cell factories”. For long-termproduction, it is desirable that the introduced nucleic acid isincorporated in the genome of the host cell, or otherwise stablymaintained. That can be readily ascertained. As indicated above, therange of proteins produced in this way is large, including enzymes forscientific and industrial use, proteins for use in therapy andprophylaxis, immunogens for use in vaccines and antigens for use indiagnosis.

[0097] The present invention provides a non-viral vector that is capableof high efficiency transfection. In a preferred embodiment, the vectorcomprises four modular elements; an oligolysine, especially [K]₁₆, DNAor RNA-binding element; a high affinity integrin-binding peptide, forexample, a peptide described herein; a DNA or RNA sequence, optionallyin a plasmid, and optionally regulated by a viral promoter and anenhancing element; the cationic liposome DOTMA/DOPE (lipofectin). Thecombination of oligolysine-peptide/DNA or RNA complex with the cationicliposome formulation DOTMA/DOPE is a potent combination. Alternatively aDOPE/DOSPA formulation may be used instead of or in addition to aDOTMA/DOPE formulation. The optimisation of variables associated withcomplex formation and the mode of transfection by LID complexes has beendemonstrated. In addition, analysis by atomic forces microscopy has beencarried out to assess the structure of the complexes.

[0098] The most important variables in the formation of optimal LIDtransfection complexes appear to be the ratio of the three componentsand their order of mixing. The same composition appears to be optimalfor all cell lines tested.

[0099] The mechanism of action of the complex of the present invention,the reason for the unexpectedly high levels of transfection and thesurprisingly wide variety of cells that can be transfected at that highefficiency are not yet understood.

[0100] However, the following observations made as a result of thepresent invention indicate that the role of the lipid component is toenhance the efficiency of transfection mediated byoligolysine-peptide/DNA or RNA complexes:

[0101] The level of transfection with LID(lipofectin/[K]₁₆-peptide/plasmid) complexes is three to six fold higherthan that with LKD (lipofectin/[K]₁₆/,plasmid) complexes prepared withthe same charge ratios, or with LD (lipofectin/plasmid) complexes. Thisindicates that the integrin-targeting moiety, i.e. the peptide, is asignificant factor in the transfection efficiency of those complexes.

[0102] Optimised LID transfection complexes contain only one seventh ofthe amount of lipofectin required for optimal transfection with LDcomplexes. Transfections with low-ratio LD complexes that contain thesame ratio of lipofectin to [K]₁₆-peptide/plasmid as in optimal LIDcomplexes but no [K]₁₆-peptide, did not transfect cells at all. Thissuggests that the role of lipofectin in LID complexes is to enhancetransfection mediated by the integrin receptor-binding peptide.

[0103] Furthermore, we have found that both LID and ID complexes bothform spherical particles of similar sizes. Optimal LD complexes,however, formed a tubular network with some tubule-associated particles,which suggests a different type of cellular interaction and transfectionmechanism from LID and ID transfections.

[0104] It is possible that condensation of plasmid DNA or RNA by theoligolysine element of the integrin-targeting oligolysine-peptides andthe cationic charge of the complexes may lead to high levels ofexpression when associated with lipofectin, and the integrin targetingmoiety i.e. the peptide is irrelevant. Transfection experiments with LKDcomplexes, mixed in the same order and the same charge ratios as the LIDcomplexes, were more efficient than LD or KD complexes. To assess thecontribution of the relative importance of the oligolysine element andthe integrin-targeting peptide domain of the combined integrin-bindingcomponent/polycationic DNA or RNA-binding component I, transfection byLID complexes were prepared containing a range of proportions of [K]₁₆and [K]₁₆integrin targeting peptide 6, [K]₁₆GACRRETAWACG[SEQ.ID.NO.:18]. Transfection expression data indicate higherefficiencies with complexes in which increasing amounts of [K]₁₆peptide6 replace [K]₁₆ and a dose-dependency on the amount ofintegrin-targetting (ligand-binding) domain i.e. peptide 6.

[0105] The ratio of components mixed together to form the optimaltransfection complex is also informative as to the possible mechanism oflipofectin mediated enhancement. The DOTMA element of lipofectin iscationic, which may enhance the activity of the complex, while DOPE mayhave the ability to destabilise the endosomal membrane (Farhood et al.,1995) enhancing endosomal release of plasmid DNA or RNA. The componentsof the LID complexes are mixed together in constant optimal ratios. Itis assumed that the particles formed also contain these elements in thesame proportions. Therefore, 3 nmol negative charge from plasmid DNA orRNA are associated with approximately 21 nmol positive charge from the[K]₁₆-peptide. Lipofectin, however, provides only a further 0.25 nmol ofpositive charge. This suggests that, contrary to expectations, theenhancing effect of lipofectin in LID complexes is not charge relatedbut may relate to the membrane destabilising effect of the DOPEcomponent.

[0106] While not limited to the following theory of the mechanism ofaction, the following model of the early stages of the transfectionprocess, which is based on the observations described herein, isproposed to explain the surprising and unexpected high efficiency oftransfection by LID complexes, which high efficiency is found in all thecell types investigated.

[0107] The complexes are formed electrostatically by random associationof lipofectin, oligolysine-peptide and plasmid DNA or RNA. The relativehigh proportion of oligolysine-peptide ensures a high proportion ofintegrin-targeting ligands per plasmid molecule. Particles are formedthat contain one or more plasmids, associated with thousands ofoligolysine-peptides and, therefore, a very high concentration ofintegrin-targeting ligands. By mixing lipofectin with theoligolysine-peptide, then adding plasmid DNA or RNA complexes are formedcontaining all three components. The particles, due to the high densityof ligands, have a high avidity for integrins on cell surfaces, bind andare internalised by a phagocytic process (Hart et al., 1994). Thevesicles fuse to form endosomes where, under acid conditions, the DOPEelement contained within the particles mediates destabilisation of theendosomal membrane and subsequent plasmid release into the cytoplasm.Phagocytosed particles lacking lipofectin are degraded in the endosomes.Particles lacking the integrin-targeting moiety are less efficient atcell binding and internalisation. Both lipofectin and the oligolysine([K]₁₆) element of the oligolysine-peptides probably contribute to theoverall efficiency of the LID complexes but the integrin-targetingcapacity of the oligolysine/peptide component appears to be importantfor optimal targeting and internalisation of the complexes.

[0108] The following non-limiting Examples illustrate the presentinvention. The Examples refer to the accompanying drawings, in which:

[0109]FIG. 1 shows the effect of different amounts of lipofectin(DOTMA:DOPE) on the enhancement of transfection of ECV304 cells using acomplex consisting of lipofectin, oligolysine-peptide 1([K]₁₆GACRGDMFGCA [SEQ.ID.NO.:19]) and plasmid pGL2.

[0110]FIG. 2 shows the effect of different amounts of lipofectin on theenhancement of transfection of A375M, COS-7 and ECV-40 cells using acomplex consisting of lipofectin, oligolysine-peptide 1([K]₁₆GACRGDMFGCA) and plasmid pGL2.

[0111]FIG. 3 shows the effect of the order of mixing the components of acomplex consisting of lipofectin (L), oligolysine-peptide 1([K]₁₆GACRGDMFGCA) (I) and plasmid pGL2 (D) on the enhancement oftransfection of ECV40 cells.

[0112]FIG. 4 shows a comparison of enhancement of transfection bylipofectin of complexes containing plasmid pGL2 and oligolysine-peptide1 ([K]₁₆GACRGDMFGCA, pep 1), or oligolysinepeptide 5 ([K]₁₆GACDCRGDCFCA[SEQ.ID.NO.:20], pep 5), or oligolysine-peptide 6 ([K]₁₆GACRRETAWACG[SEQ.ID.NO.:21], pep 6) or [K]₁₆ (K16), with lipofectin (lip) andwithout lipofectin, and a complex containing plasmid pGL2 withlipofectin:[K]₁₆lysine-peptide 1 in a ratio by weight of 4:1 (Lipo 4 to1).

[0113]FIG. 5 shows the dose-dependency of a complex containinglipofectin, oligolysine-peptide 6 ([K]₁₆GACRRETAWACG) and plasmid pGL2on the availability of integrin-binding ligands.

[0114]FIG. 6 shows the structure of various complexes, as determinedusing atomic force microscopy, the complexes being formed with differentcombinations of plasmid DNA (plasmid pGL2), oligolysine-peptide([K]₁₆-peptide 6) and lipofectin as follows: A: [K]₁₆-peptide 6 andplasmid pGL2; B: [K]₁₆-peptide 6, lipofectin and plasmid pGL2; C:lipofectin and plasmid pGL2, optimal ratio; D: lipofectin and plasmidpGL2, suboptimal ratio.

[0115]FIG. 7 shows levels of expression of IL-12 48 hours aftertransfection of COS-7 cells and neuroblastoma cells lines IMR-32, KELLYand SHSY-5Y with a complex containing lipofectin, oligolysine-peptide 6([K]₁₆GACRRETAWACG) and either two retroviral plasmid constructsencoding the two domains of IL-12 (MFGS-IL12) or one plasmid containinga fusion gene, Flexi-12 under a CMV promoter.

[0116]FIG. 8 shows the effect of transfection with anti-senseoligonucleotides (AS) to the thrombin receptor (PAR-1) on thrombininduced proliferation of human foetal lung fibroblasts (HFL-1 cells).

[0117]FIG. 9 shows the effect of transfection of haematopoietic celllines HL60, PLB985, TF1 and U937 with LID complexes containinglipofectin, the reporter gene pEGFP-N1 and either [K]₁₆-peptide 6 (pep6) or [K]₁₆-peptide 8 (GGCRGDMFGCA [SEQ.ID.NO.:8] pep 8) compared withuntreated cells. The percentage of GFP positive cells is determinedusing a fluorescence activated cell sorter.

EXAMPLES MATERIALS & METHODS Cell Lines

[0118] The cell line COS-7 (monkey kidney epithelial cells) weremaintained in Dulbecco's Modified Eagle Medium (DMEM; Life Technologies,Paisley, U.K.) supplemented with 10% foetal calf serum (FCS),L-glutamine, penicillin and streptomycin. ECV304 (spontaneouslytransformed human umbilical vein endothelial cells) were grown in 199Medium (Life Technologies, Paisley, U.K.). HT1080 fibrosarcoma cells andA375M melanoma cells were maintained in DMEM and 10% FCS. IMR2neuroblastoma cells were grown in DMEM F12 Nutrient Mix (Lifetechnologies). Cell lines were all grown in a 37° C. incubator with a 5%CO₂ water-saturated atmosphere.

Peptide Synthesis

[0119] The sequence of peptide 6, GACRRETAWACG, was based on anα5β1-specific peptide from a phage display library (Koivunen et al.,1995). The oligolysine-peptide [K]₁₆GACRRETAWACG was synthesised asfollows:

[0120] Protected amino acids and preloaded Gly-Wang resin were obtainedfrom Calbiochem-Novabiochem (Nottingham, U.K.). Solvents and HBTU[2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate] were obtained from Perkin-Elmer Applied Biosystems,U.K. The peptide was synthesised on a Model 431A updated AppliedBiosystems Solid Phase Synthesiser on 0.1 mmol preloaded Gly-Wang resin(Calbiochem-Novabiochem, Nottingham, U.K.) using basic feedbackmonitoring cycles and HBTU as a coupling reagent.9-fluorenylmethyloxy-carbonyl was used for temporary α-amino groupprotection. Side-chain protecting groups were tert-butyloxycarbonyl forLys and Trp, trityl for Cys, 2,2,5,7,8-pentamethylchroman-6sulphonyl forArg, tert-butylester for Glu and tert-butyl ether for Thr. Cleavage fromthe resin and deprotection of the peptide was achieved by treating thepeptidyl-resin with 10 ml of a mixture containing 10 ml trifluoroaceticacid, 0.25 ml ethanedithiol, 0.25 ml triisopropylsilane at 20° C. fortwo hours. The peptide was precipitated using ice-cold diethylether andthen filtered through a fine sintered glass filter funnel under lightvacuum. The peptide precipitate was dissolved in 10% acetic acid/watersolution and freeze dried. The crude peptide was analysed by reversephase HPLC and matrix assisted laser desorption ionisation time offlight mass spectroscopy. Purity of the crude peptide was about 70% byreverse phase HPLC, and mass analysis using a Finnegan LazerMat gave amolecular weight of 3331.5 for the MH+ ion which was in excellentagreement with calculated weight for MH+ ion of 3331.46.

[0121] Oligolysine-peptide 1: [K]₁₆GACRGDMFGCA and oligolysine-peptide5: [K]₁₆GACDCRGDCFCA were obtained from Zinsser Analytic (Maidenhead,U.K.).

Plasmid DNA

[0122] The plasmids pGL2, which contains a luciferase reporter gene(Promega, Madison, Wis., U.S.A.) and PCMVB, which contains aβ-galactosidase reporter gene (Clontech, Palo Alto, Calif., U.S.A.) weregrown in Escherichia coli DH5 a and purified, after bacterial alkalinelysis, on Qiagen resin columns (Qiagen Ltd., Crawley, U.K.) by themanufacturer's instructions. Isopropanol-precipitated DNA pellets werewashed with 70% ethanol then dissolved in water or TE buffer (10 mMTris-Cl, pH 8.0 and 1 mM EDTA).

[0123] Spectrophotometric measurements of plasmid solutions were used toassess plasmid concentration (A₂₆₀) and purity (A₂₆₀/A₂₈₀ ratio).Plasmid solutions were adjusted to a concentration of 1 mg/ml and storedat 4° C.

Formation of Transfection Complexes

[0124] Cells were seeded into 24-well plates at 5×10⁴ cells per wellthen incubated overnight at 37° C. in complete growth medium. Thefollowing day, transfection complexes were made from the following stocksolutions, all prepared in OptiMEM (Life Technologies, Paisley, U.K.),lipofectin (an equimolar mixture of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE), obtainedas “Lipofectin” from Life Technologies, Paisley, U.K.) (1 mg /ml),pGL2-control (1 mg/100 ml) and [K]₁₆/integrin-targeting peptide 1, 5 or6 (0.1 mg/ml).

[0125] Complexes were made usually with three components:oligolysine-peptide (I), plasmid DNA or RNA (D) and lipofectin (L), bymixing together the different components with an automatic pipette. Themixture LID was made in the same way in the optimal weight ratio0.75:4:1 (L:I:D). Both types of mixture were left to aggregate for atleast 30 min then diluted to a concentration of one microgram DNA per0.5 ml with OptiMEM. The growth medium was removed from each well then0.5 ml of transfection complex added. The plate was then returned to theincubator for four to six hours. The transfection medium was thenremoved and replaced with 1 ml of complete growth medium. Transfectedcells were incubated for 48 to 72 hours then assayed for reporter geneactivity.

Luciferase Assays

[0126] Cells transformed with pGL2 were washed twice with PBS to removeserum then 100 microliters of Reporter Lysis Buffer (Promega, Madison,Wis., U.S.A.) was added to each well and placed at 40° C. for 15 to 30minutes. Cells were then dislodged by scraping with a yellowmicropipette tip. Cellfree lysates were then prepared and assayed with aLuciferase Assay kit (Promega, Madison, Wiss., U.S.A.) following themanufacturer's instructions. Total light emission was measured for 60seconds on an LKB 1251 Luminometer (Labtech, Uckfield, U.K.). Theprotein concentration of each sample was then determined with ProteinAssay Reagent (BioRad, Hercules, Calif., U.S.A.) and luciferase enzymeactivity expressed in terms of relative light units per milligram ofprotein (RLU/mg).

LacZ Assays

[0127] β-galactosidase activity was detected by staining with X-gal.After washing with PBS cells were fixed to the plastic plates byaddition of 0.5% glutaraldehyde in PBS for 20 minutes at 40° C. Wellswere washed with PBS and cells were stained with X-gal at 370 C. for upto six hours.

Atomic Forces Microscopy (AFM)

[0128] Atomic forces microscope analysis of transfection complexes wasperformed as described previously (Wolfert & Seymour, 1996) using anAFM-2, part of the NanoScope II (Digital Instruments, Santa, Barbara,U.S.A.). Transfection complexes of [K]₁₆-peptide 6/pGL2, with andwithout lipofectin, were prepared as described above except that waterwas used as the diluent for all components rather than optiMEM.

Example 1: Effect of Different Amounts of Lipofectin (DOTHA/DOPE) onTransfection

[0129] Transfection complexes were prepared as described above in theMaterials & Methods section. The complexes were made by mixing solutionsof oligolysine-peptide 1 ([K]₁₆GACRGDMFGCA) at 0.1 mg/ml in OptiMEM lowserum tissue culture medium with a solution of lipofectin (DOTMA/DOPEcationic liposome as above) in a range of concentrations from 1 to 10μg/100 μl in OptiMEM. Finally, the appropriate amount of pGL2-controlplasmid DNA (0.1 mg/ml) was added and mixed by repeated pipetting. Theratio of mixing of each component was a constant 4 μg ofoligolysine-peptide per μg of DNA, while the proportion of lipofectinvaried from 1 to 10 μg per μg of DNA. ECV304 cells were transfected withthe complexes as described above, incubated for 48 hours then assayedfor luciferase expression as described above. The results are shown inFIG. 1.

[0130] Complexes formed with 1 μg of lipofectin and 4 μg ofoligolysine-peptide per microgram of plasmid were almost 100-fold moreactive than complexes lacking lipofectin. Addition of larger amounts oflipofectin reduced transfection activity in a lipofectin dose-dependentmanner.

[0131] Similar results were obtained with [K]₁₆-peptide 6.

Example 2: Effect of Different Amounts of Lipofectin on Transformationin Three Different Cell Lines

[0132] Experiments were then performed to refine the optimal amount oflipofectin in LID transfection complexes using three different celllines A375M (melanoma cells), COS-7 (monkey kidney epithelial cells) andECV304 (human umbilical cord endothelial cells).

[0133] Transfection complexes were made as described in Example 1 butusing a narrower range of amounts of lipofectin.Lipofectin/oligolysine-peptide/DNA complexes were prepared with constantamounts of [K]₁₆-peptide 1 ([K]₁₆GACRGDMFGCA) (4 μg) and pGL2 (1 μg)plasmid DNA and a range of lipofectin amounts (1 to 2.5 micrograms).Complexes were used to transfect A375M, COS-7 and ECV304 cells, whichwere then harvested two days later for luciferase expression analysis.

[0134] The results are shown in FIG. 2. In each case the optimaltransfection ratio peaked at 0.75 μg of lipofectin per microgram ofplasmid DNA. This combination of the amounts of the components wasmaintained in all subsequent examples.

[0135] A mixing ratio L:I:D of 0.75:4:1 by weight corresponds to a molarratio of 0.5 nmol lipofectin: 1.25 nmol oligolysine-peptide 1: 0.25 pmolpGL2-control. The molar charge of each component is 0.5 moles positivecharge per mole lipofectin, seventeen moles positive charge,per mole[K]₁₆-peptide 1 and 12,000 moles negative charge per mole of pGL2 (6kb). Therefore, in the optimal transfection complex, 3 nmol of negativecharge from the plasmid is mixed with 21 nmol of positive charge fromoligolysine-peptide 1 and 0.25 nmol positive charge from lipofectin.Hence the charge ratio of approximately 7:1 positive to negative chargesin ID complexes is little altered by the incorporation of 0.25 nmolpositive charge from lipofectin into high efficiency LID transfectioncomplexes. It is likely, therefore, that the improvement in transfectionefficiency of LID complexes is not charge related.

Example 3: Effect of the Order in Which the Components of the Complexare Mixed

[0136] To determine the procedure for the production of optimal LIDtransfection complexes transfections were performed with complexes madeby adding the components of the complexes in different orders. Allcombinations were prepared with the same amounts and concentrations ofthe components (1 μg pGL2 plasmid DNA, 0.75 μg of lipofectin and 4 μg ofoligolysine-peptide 1 ([K]₁₆GACRGDMFGCA). Transfections were performedin ECV304 cells and luciferase activity was assessed as described above.

[0137] The results are shown in FIG. 3 in which D represents the plasmidvector pGL2, I represents [K]₁₆-peptide 1 and L represents lipofectin.The expression data indicates that the order of mixing LID was optimal.Significantly, combinations in which the lipofectin was the lastcomponent added were least efficient. The order of mixing, LID, wasemployed in all subsequent transfection experiments.

Example 4: Transfection Rates

[0138] Cells were transfected with optimisedoligolysine-peptide/lipofectin/pCMVβ complexes as described in Examples1 and 2 prepared in the order of mixing LID but using PCMVβ as theplasmid vector (component D) instead of pGL2. The cells were stained forβ-galactosidase activity with X-qal as described above. A number of celltypes, A375M, COS-7 and ECV304 displayed transfection efficiencies of 50to 100% compared to 1 to 10% achieved with oligolysine-peptide/DNAcomplexes alone. This represents a very significant improvement intransfection efficiency.

Example 5: Comparison of Enhancement with Lipofectin and with DifferentOligolysine-peptides

[0139] To compare the effect of different integrin-targetingoligolysine-peptides, duplicate sets of complexes were formed withplasmid pGL2 and one of the following:

[0140] oligolysine-peptide 1 ([K]₁₆GACRGDMFGCA, pep 1),

[0141] oligolysine-peptide 5 ([K]₁₆GACDCRGDCFCA, pep 5),

[0142] oligolysine-peptide 6 ([K]₁₆GACRRETAWACG, pep 6), and [K]₁₆.

[0143] One set of complexes also contained lipofectin (lip), the otherwas without lipofectin. A control complex containing plasmid pGL2 withlipofectin and [K]₁₆lysine-peptide 1 in a ratio by weight of 4:1 wasprepared.

[0144] Each complex was used to transfect cell lines and luciferaseexpression determined. Complexes were made with (lip) and withoutlipofectin. An optimised complex was performed for comparison. Alloligolysine-peptides were mixed with lipofectin and plasmid DNA (KLD) inthe same optimised charge ratios and order of mixing.

[0145] The results are shown in FIG. 4. Although KLD complexes wereusually better transfection agents than KD or LD complexes, LIDcomplexes generated luciferase expression levels three to six-foldhigher than KLD complexes. Expression levels from LID complexescontaining oligolysine-peptide 5 were two-fold lower than thosecontaining oligolysine-peptide 1 or oligolysine-peptide 6, which mayreflect the differing integrin receptor affinities of the peptides. Thetransfection enhancement of the LID complexes was observed with all thepeptides tested, two of which (peptides 1 and 5) contain the conservedRGD sequence, one of which (peptide 6) does not.

Example 6: Specificity

[0146] To demonstrate integrin specificity, LID complexes were preparedwith constant amounts of plasmid pGL2-control and lipofectin, and arange of combinations of [K]₁₆-peptide 6 and [K]₁₆. A total of 40 μg of[K]₁₆-peptide was used, consisting of 1, 5, 10, 20, 35, 39 μg of[K]₁₆-peptide 6 made up to 40 μg with [K]₁₆.

[0147] Transfections were performed as described in Example 1 andluciferase assays performed after 48 hours. The results are shown inFIG. 5. Transfection efficiency demonstrated an apparently exponentialincrease with increasing amounts of oligolysine-peptide 6, and,therefore, a dose-dependent response to the amount of availableintegrin-binding ligands. Accordingly, while both the sixteen-lysinedomain, and the lipofectin components are themselves capable ofmediating transfection, both individually and in [K]₁₆/lipofectincombination complexes, the highest efficiency transfection is directlyproportional to the amount of available integrin-binding ligand.

Example 7: Atomic Force Microscopy

[0148] Atomic force microscopy experiments were performed to determineand compare the structures formed by mixing 4 μg [K]₁₆peptide 6 and 1 μgpGL2-control plasmid DNA (ID complexes). LID complexes were formed from[K]₁₆-peptide 6 (4 μg)/lipofectin (0.75 μg)/DNA (1 μg) in the order LIDwhich was shown to yield optimal transfection results. Lipofectin/DNAcomplexes (LD) were formed at two different ratios; an optimaltransfection ratio of 5 μg lipofectin per microgram of pGL2 and the sameratio as used in LID complexes, 0.75 μg lipofectin per microgram ofplasmid.

[0149] The results are shown in FIG. 6. ID complexes, composed ofoligolysine-peptide 6 and plasmid DNA, were examined initially by AFMwithin fifteen minutes of mixing the two components. The complexesformed particles of low polydispersity which, on the mica coverslips,had a diameter of approximately 200 nm. A computer-generated contour maprevealed that the particles formed were of irregular conical shape. LIDcomplexes assessed by AFM formed particles of a similar size and shapeto ID complexes. The additional lipofectin did not, apparently, disruptthe particles. LD complexes, however, formed at the 5:1 ratio appearedas a network of tubes with occasional particles associated with thetubes. LD complexes formed at the lower ratio (0.75:1), however,appeared to be short tubular structures. LD complexes formed at thislower ratio were inactive in transfection experiments. LID complexesformed as above were also analysed by AFM after standing overnight.Particles were now smaller in size with diameters of approximately50-100 nm suggesting that the particles had compacted.Computer-generated computer maps represented these particles as regularconical structures. The cones were measured and their volumes werecalculated. The spheres which the particles are predicted to form whenfree in solution were then calculated to be 20 to 60 nm in diameter. Intransfection experiments with pGL2 the compact particles formedovernight in water yielded luciferase expression results approximatelytwice as high as the freshly made complexes.

Example 8: Transfection of Neuroblastoma Cells

[0150] Transfection of three different human neuroblastoma cell lines,SHSY-5Y, KELLY and IMR-32 and one mouse neuroblastoma cell line, Nb2A,was optimised using an LID complex containing [K]₁₆-peptide 6,lipofectin and either luciferase or GFP as reporter gene, as describedin the Materials and Methods section and the Examples above.

[0151] The three human neuroblastoma cell lines and COS-7 cells werethen transfected using the same LID complex with, instead of thereporter gene, one of two different IL-12 expressing vectors. One vectorexpresses a fusion protein of the two chains of IL-12, p35 and p40,(Flexi-12; Anderson et al. 1997) This fusion is regulated by a CIVpromoter. The second IL-12 expression system consists of two retroviralconstructs MFGS-p35 and MFGS-p40, which are retroviral plasmidconstructs encoding the two separate chains of interleukin-12 (IL-12).Both genes are regulated by the retroviral long terminal repeats (LTRs).The vectors were obtained from Professor Mary Collins, UCL, London.

[0152] Secreted IL-12 expression was monitored by ELISA 48 hours aftertransfection. The transfected cells were found to secrete high levels ofthe cytokine, see FIG. 3. The Flexi-12 construct was most efficient.

[0153] These results demonstrate that the transfection system of thepresent invention is suitable for use in a vaccine for neuroblastoma, animportant childhood malignancy, and also for vaccines against othercancers.

Example 9: Transfection of Lung Bronchial Epithelium In Vivo

[0154] LID complexes comprising [K]₁₆-peptide 6 and lipofectin in theoptimal ratio L:I:D of 0.75:4:1 mixed in the order LID were made asdescribed in the Materials and Methods section above but using asolution of the oligolysine-peptide in phosphate buffered saline (PBS)at a concentration of 1 mg/ml. The other components were in solution inwater, lipofectin at a concentration of 1 mg/ml and DNA encoding anuclear localising betagalactosidase reporter gene pAB11 at aconcentration of 1 mg/ml. The oligolysine-peptide was used at highconcentration to minimise the final volume of the complex, and PBS wasused instead of OptiMEM for bio-compatibility.

[0155] Lewis rats were anaesthetized and then injected through thetrachea into the airway with 287.5 μl of complex comprising 37.5 1lipofectin, 200 μg [K]₁₆-peptide 6 in 200 μl PBS, and 50 μg pAB11 in 50μl water. The animals were sacrificed at 24 hours, the lungs removed,fixed and stained with X-gal, then sectioned and examined. Extensivestaining was seen in the bronchial epithelium in the upper airway.

[0156] This result demonstrates the utility of the transfection complexof the present invention for gene therapy of disease involving the lungsand airways, for example, cystic fibrosis and asthma.

Example 10: Transfection of Corneal Endothelium In Vivo

[0157] LID complexes were made as described in Example 10 for in vivolung transfections. The LID complex-containing solution was injectedinto the anterior chamber of the eye of mice. The volume of solutioninjected in each case was 2 μl, thus delivering approximately 0.2 μg ofpAB11 plasmid DNA. Efficient gene transfer to the corneal endotheliumwas demonstrated by X-gal staining.

[0158] The high transfection rate demonstrates the utility of thetransfection complex of the invention of the treatment of eye diseasesaffecting the cornea, and for corneal transplantation.

Example 11: Transfections of Primary Smooth Muscle Cells and CardiacMyocytes

[0159] Tissue cultures of rat primary smooth muscle cells (aortic smoothmuscle cells) and cardiac myocytes were prepared according to standardmethods (Blank et al. 1988). An LID complex comprising lipofectin,[K]₁₆-peptide 6 and GFP as a reporter gene in the optimal LID ratio andmixing order was prepared as described in the Materials and Methodssection and the Examples above. The tissue cultures were transfectedwith the LID complex as described in the Material and Methods sectionabove. Fluorescing imaging of GFP-expressing cells demonstratestransfection efficiency in excess of 50%.

[0160] Primary smooth muscle cells and cardiac myocytes are particularlyresistant to plasmid-mediated transfection using most other non-viralvectors. In contrast, the transfection complex of the present inventionachieved transfection efficiencies in excess of 50%, thus demonstratingthe utility of the complexes for treatment of diseases affecting muscle,including smooth muscle and cardiac muscle.

Example 12: Transfections with High Molecular Weight Constructs

[0161] Different size constructs can be delivered with the transfectioncomplex of the present invention. A fibroblast culture was transfectedas described in the Materials and Methods section with an LID complexcomprising [K]₁₆-peptide 6, lipofectin and a 130 kB DNA construct. Thecomplex, comprising the LID components in the optimal ratio and mixingorder, was prepared as described in the Methods and Materials sectionand Examples above. Transfection was achieved with 2-3% efficiency.Cellular process associated with the enhanced integrin-mediatedinternalisation of DNA using a complex of the present invention are moreclosely related to phagocytosis than endocytosis and are thusparticularly suited to the delivery of complexes containing very largeDNA molecules.

Example 13: Transfection with Anti-sense DNA

[0162] Thrombin stimulates proliferation of human lung fibroblasts.Thrombin-treated human lung fibroblasts (HFL-1 cells) proliferated 53%in response to thrombin. 24 hours before treatment with thrombin, HFL-1cells were treated with an LID complex comprising [K]₁₆-peptide 6,lipofectin and a 20-mer antisense oligonucleotide directed against thethrombin receptor PAR-1 in the optimal ratio and mixing order preparedas described in the Materials and Methods section and the Examplesabove. The antisense oligonucleotide-containing complex was in contactwith the cells for 4 hours. 24 hours after the start of the treatmentwith the complex, treatment with thrombin was carried out.

[0163] The thrombin-induced proliferation was attenuated by 76% +/−12%by the pre-treatment with the LID complex. Cells treated with theantisense-containing complex but not with thrombin did not proliferate.

[0164] This result demonstrates the utility of the complex of theinvention for efficient intracellular transport of antisenseoligonucleotides, as is required for antisense therapy, for example,antiviral and anticancer therapy.

Example 14: Transfection of Haematopoietic Cells

[0165] Haematopoietic cells are particularly resistant to transfectionwith most plasmid-mediated vectors.

[0166] LID complexes were prepared as described in the Material andMethods section and Examples above using lipofectin and [K]₁₆-peptide 6,which targets α5β1 integrins, and pEGFP-N1 (Promega) as reporter gene.Complexes were prepared analogously substituting [K]₁₆-peptide 8([K]₁₆GACQIDSPCA SEQ.ID.NO.:21), which targets α4β1 integrins, for[K]₁₆-peptide 6. The complexes were prepared by mixing the components inthe optimal ratio and mixing order as described in the Materials andMethods section and Examples above.

[0167] Four different haematopoietic cells lines (HL60, PLB985, TF1 andU937) were transfected as described in the Materials and Methods sectionwith the following modifications: cells were untreated or were treatedwith Gm-CSF (10 ng/ml) for TF1 cells or phorbol myristic acid (PMA) forthe other three cells lines prior to transfection. Transfection with theLID complexes containing pEGFP-N1 generated a transfection efficiency ofmore than 60% in all four lines as measured on fluorescent activatedcell sorter, see FIG. 8.

[0168] These results demonstrate the utility of the transfection complexof the invention for gene therapy involving haematopoietic cells, forexample, gene therapy of leukaemia and bone marrow stem cell disorders.This is particularly useful because, as pointed out above,haematopoietic cells are particularly resistant to transfection withmost plasmid-mediated vectors.

REFERENCES

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[0170] 2. Wagner E, Cotten M, Mechtler K, Kirlappos H, Birnstiel M L.DNA-binding transferrin conjugates as functional gene-delivery agents:Synthesis by linkage of polylysine or ethidium bromide to thetransferrin carbohydrate moiety. Bioconjugate Chemistry 1991;2:226-231.

[0171] 3. Cotten M, Lange. Transferrin-polycation-mediated introductionof DNA into human leukemic cells: Stimulation by agents that affect thesurvival of transfected DNA or modulate transferrin receptor levels.PNAS 1990;87:4033-4037.

[0172] 4. Ferkol T, Perales J C, Eckman E, Kaetzel C S, Hanson R W,Davis P B. Gene transfer into the airway epithelium of animals bytargeting the polymeric immunoglobulin receptor. J ClinicalInvestigation 1995;95:493-502.

[0173] 5. Curiel D T, Agarwal S, Wagner E, Cotten M. Adenovirusenhancement of transferrin-polylysine-mediated gene delivery. PNAS1991;88:8850-8854.

[0174] 6. Fernandez M A, Muno-Fernandez M A, Fresno M. Involvement of β1integrins in the binding and entry of Trypanosoma cruzi into humanmacrophages. European J of Immunology 1993;23:552-557.

[0175] 7. Wickham T J, Filardo E J, Cheresh D A, Nemerow G R. Integrinαvβ5 selectively promotes adenovirus mediated cell membranepermeabilization. J Cell Biology 1994;127(1):257-264.

[0176] 8. Bergelson J M, Shepley M P, Chan B M C, Hemler M E, Finberg RW. identification of the integrin VLA-2 as a receptor for echovirus 1.Science 1992;255:1718-1720.

[0177] 9. Logan D, Abu-Ghazaleh R, Blakemore W, et al. Structure of amajor immunogenic site on foot-and-mouth disease virus. Nature1993;362:566-568.

[0178] 10. Isberg R R. Discrimination between intracellular uptake andsurface adhesion of bacterial pathogens. Science 1991;252:934-938.

[0179] 11. Almeida E A C, Huovilla A-P J, Sutherland A E, et al. Mouseegg integrin α6β1 functions as a sperm receptor. Cell 1995;81:1095-1104.

[0180] 12. Clements J M, Newham P, Shepherd M, et al. Identification ofa key integrin-binding sequence in VCAM-1 homologous to the LDV activesite in fibronectin. J Cell Science 1994;107:2127-2135.

[0181] 13. Lu X, Deadman J J, Williams J A, Kakkar V V, Rahman S.Synthetic RGD peptides derived from the adhesive domains of snake-venomproteins: evaluation as inhibitors of platelet aggregation. BiochemistryJ 1993;296:21-24.

[0182] 14. Koivunen E, Wang B, Ruoslahti E. Phage libraries displayingcyclic peptides with different ring sizes: ligand specificities of theRGD-directed integrins. Biol/Technology 1995;13:265-270.

[0183] 15. Koivunen E, Gay D A, Ruoslahti E. Selection of peptidesbinding to the α5β1 integrin from phage display library. J BiologicalChemistry 1993;268(27):20205-20210.

[0184] 16. Koivunen E, Wang B, Ruoslahti E. Isolation of a highlyspecific ligand for the α5β1 integrin from a phage display library. JCell Biology 1994;124(3):373-380.

[0185] 17. O'Neil K T, Hoess R H, Jackson A, Ramachandran N S, Mousa A,DeGrado W F. Identification of novel peptide antagonists for GPIIb/IIIafrom a conformationally constrained phage peptide library. Proteins1992;14:509-515.

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[0188] 20. Hart S L, Knight A M, Harbottle R P, et al. Cell binding andinternalization by filamentous phage displaying a cyclicArg-Gly-Asp-containing peptide. J Biological Chemistry1994;269:12468-12474.

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1 16 28 amino acids amino acid circular peptide 1 Gly Gly Cys Arg GlyAsp Met Phe Gly Cys Gly Gly Lys Lys Lys Lys 1 5 10 15 Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys 20 25 27 amino acids amino acid circularpeptide 2 Gly Gly Cys Arg Gly Asp Met Phe Gly Cys Gly Lys Lys Lys LysLys 1 5 10 15 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 20 25 26 aminoacids amino acid circular peptide 3 Gly Gly Cys Arg Gly Asp Met Phe GlyCys Lys Lys Lys Lys Lys Lys 1 5 10 15 Lys Lys Lys Lys Lys Lys Lys LysLys Lys 20 25 27 amino acids amino acid circular peptide 4 Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Gly AlaCys Arg Gly Asp Met Phe Gly Cys Ala 20 25 8 amino acids amino acidcircular peptide 5 Cys Arg Gly Asp Met Phe Gly Cys 1 5 10 amino acidsamino acid circular peptide 6 Gly Gly Cys Arg Gly Asp Met Phe Gly Cys 15 10 11 amino acids amino acid circular peptide 7 Gly Gly Cys Arg GlyAsp Met Phe Gly Cys Gly 1 5 10 11 amino acids amino acid circularpeptide 8 Gly Gly Cys Arg Gly Asp Met Phe Gly Cys Ala 1 5 10 11 aminoacids amino acid circular peptide 9 Gly Ala Cys Arg Gly Asp Met Phe GlyCys Ala 1 5 10 12 amino acids amino acid circular peptide 10 Gly Ala CysAsp Cys Arg Gly Asp Cys Phe Cys Ala 1 5 10 12 amino acids amino acidcircular peptide 11 Gly Ala Cys Arg Arg Glu Thr Ala Trp Ala Cys Ala 1 510 12 amino acids amino acid linear peptide 12 Gly Ala Cys Arg Arg GluThr Ala Trp Ala Cys Gly 1 5 10 9 amino acids amino acid circular peptide13 Cys Arg Arg Glu Thr Ala Trp Ala Cys 1 5 12 amino acids amino acidlinear peptide 14 Gly Ala Gly Pro Glu Ile Leu Asp Val Pro Ser Thr 1 5 1010 amino acids amino acid circular peptide 15 Gly Ala Cys Gln Ile AspSer Pro Cys Ala 1 5 10 25 amino acids amino acid circular peptide 16 GlyAla Cys Arg Arg Glu Thr Ala Trp Ala Cys Gly Lys Gly Ala Cys 1 5 10 15Arg Arg Glu Thr Ala Trp Ala Cys Gly 20 25

1. A complex that comprises (i) a nucleic acid, (ii) an integrin-bindingcomponent, (iii) a polycationic nucleic acid-binding component, and (iv)a lipid component
 2. A complex as claimed in claim 1, wherein theintegrin-binding component is an integrin-binding peptide
 3. A complexas claimed in claim 2, wherein the peptide consists of or comprises allor part of the integrin-binding domain of a naturally-occurring integrinligand.
 4. A complex as claimed in claim 3, wherein the integrin-bindingpeptide comprises the conserved amino acid sequencearginine-glycine-aspartic acid (RGD).
 5. A complex as claimed in claim4, wherein a peptide comprising the sequence RGD has a cyclic region inwhich the conformational freedom of the RGD sequence is restricted.
 6. Acomplex as claimed in claim 5, wherein a cyclic peptide has two or morecysteine residues that form one or more disulphide bond(s).
 7. A complexas claimed in claim 6, wherein the peptide consists of or comprises thesequence CRGDMFGC [SEQ.ID.NO.:5].
 8. A complex as claimed in claim 7,wherein the peptide consists of or comprises the sequence GGCRGDMFGC[SEQ.ID.NO.:6], GGCRGDMFGCG [SEQ.ID.NO.7], GGCRGDMFGCA [SEQ.ID.NO.:8] orGACRGDMFGCA [SEQ.ID.NO.:9].
 9. A peptide as claimed in claim 6, whichpeptide consists of or comprises the sequence GACDCRGDCFCA[SEQ.ID.NO.:10].
 10. A peptide as claimed in claim 2, which peptideconsists of or comprises the sequence CRRETAWAC [SEQ.ID.NO.:13].
 11. Apeptide as claimed in claim 10, which consists of or comprises thesequence GACRRETAWACA [SEQ.ID.NO.:11] or GACRRETAWACG [SEQ.ID.NO.:12].12. A peptide as claimed in claim 2, which consists of or comprises thesequence GAGPEILDVPST [SEQ.ID.NO.:14], GACQIDSPCA [SEQ.ID.NO.:15] orGACRRETAWACGKGACRRETAWACG [SEQ.ID.NO.:16].
 13. A complex as claimed inany one of claims 1 to 12, wherein the nucleic acid component is orrelates to a gene that is the target for gene therapy, gene vaccinationor anti-sense therapy.
 14. A complex as claimed in any one of claims 1to 13, wherein transcriptional and/or translational control elements forthe nucleic acid are provided and the nucleic acid is optionally packedin a phage or vector.
 15. A complex as claimed in any one of claims 1 to14, wherein the nucleic acid component is DNA.
 16. A complex as claimedin any one of claims 1 to 14, wherein the nucleic acid component is RNA.17. A complex as claimed in any one of claims 1 to 16, wherein thenucleic acid-binding component has from 3 to 100 cationic monomers. 18.A complex as claimed in any one of claims 1 to 17, wherein thepolycationic nucleic acid-binding component is an oligolysine.
 19. Acomplex as claimed in claim 18, wherein the oligolysine has from 10 to20, especially 16 lysine residues.
 20. A complex as claimed in any oneof claims 1 to 19, wherein the lipid component is or is capable offorming a cationic liposome.
 21. A complex as claimed in any one ofclaims 1 to 20, wherein the lipid component is or comprises one or morelipids selected from cationic lipids and lipids having membranedestabilising or fusogenic properties.
 22. A complex as claimed in claim21, wherein the lipid component is or comprises the neutral lipiddioleyl phosphatidylethanolamine (DOPE) or a lipid having similarmembrane destabilising or fusogenic properties.
 23. A complex as claimedin claim 21 or claim 22, wherein the lipid component is or comprises thecationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride (DOTMA) or a lipid having similar cationic properties.
 24. Acomplex as claimed in claim 23, wherein the lipid component is orcomprises a mixture of DOPE and DOTMA, especially an equimolar mixturethereof.
 25. A complex as claimed in claim 24, which comprises anequimolar mixture of DOPE and DOTMA as the lipid component, anintegrin-binding peptide as the integrin-binding component, and [K]₁₆ asthe polycationic nucleic acid-binding component.
 26. A complex asclaimed in claim 24 or claim 25, wherein the ratio lipidcomponent:integrin-binding/polycationic nucleic acid-binding component:nucleic acid is 0.75:4:1 by weight or 0.5 nmol:1.25 nmol:0.25 nmol on amolar basis.
 27. A complex as claimed in any one of claims 1 to 24,wherein the lipid component is or comprises2,3-dioleyloxy-N-[2-(spermidinecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium-trifluoridoacetate(DOSPA) or a lipid having similar properties to those of DOSPA.
 28. Acomplex as claimed in claim 27, wherein the lipid component is orcomprises a mixture of DOPE and DOSPA, especially a mixture of one partby weight DOPE to 3 parts by weight DOSPA.
 29. A complex as claimed inclaim 28, which comprises a mixture of DOPE and DOSPA as the lipidcomponent, an integrin-binding peptide as the integrin-bindingcomponent, and [K]₁₆ as the polycationic nucleic acid-binding component.30. A complex as claimed in claim 29, wherein the ratio lipidcomponent:polycationic nucleic acid-binding component: nucleic acid is12:4:1 by weight.
 31. A process for the production of a complex asclaimed in any one of claims 1 to 30, which comprises admixingcomponents (i), (ii), (iii) and (iv).
 32. A process as claimed in claim31, wherein the components are admixed in the following order: lipidcomponent, integrin-binding component/polycationic nucleic acid-bindingcomponent, nucleic acid.
 33. A complex as claimed in any one of claims 1to 30, obtainable by a process as claimed in claim 31 or claim
 32. 34. Amixture comprising an integrin-binding component, a polycationic nucleicacid-binding component, and a lipid component.
 35. A mixture as claimedin claim 34, wherein the integrin-binding component is as defined in anyone of claims 2 to
 12. 36. A mixture as claimed in claim 34 or claim 35,wherein the polycationic nucleic acid-binding component is as defined inany one of claims 17 to
 19. 37. A mixture as claimed in any one ofclaims 34 to 36, wherein the lipid component is as defined in any one ofclaims 20 to 24, 27 and
 28. 38. A mixture as claimed in claim 34, whichcomprises an equimolar mixture of DOPE and DOTMA as the lipid component,an integrin-binding peptide as the integrin-binding component, and [K]₁₆as the polycationic component nucleic acid-binding component.
 39. Amixture as claimed in claim 38, wherein the ratio lipidcomponent:combined integrin-binding/polycationic nucleic acid-bindingcomponent is 0.75:4 by weight.
 40. A process for producing a complex asclaimed in claim 1, which comprises incorporating a nucleic acid with amixture as claimed in any one of claims 34 to
 39. 41. A method oftransfecting a cell with a nucleic acid, which comprises contacting thecell in vitro or in vivo with a complex as claimed in any one of claims1 to 30 or claim
 33. 42. A pharmaceutical composition which comprises acomplex as claimed in any one of claims 1 to 30 claim 33, in admixtureor conjunction with a pharmaceutically suitable carrier.
 43. A methodfor the treatment or prophylaxis of a condition caused in human or or ina non-human animal by a defect and/or a deficiency in a gene, whichcomprises administering a complex as claimed in any one of claims 1 to30 or claim 33 to the human or to the non-human animal.
 44. A method fortherapeutic or prophylactic immunisation of a human or of a non-humananimal, which comprises administering a complex as claimed in any one ofclaims 1 to 30 or claim 33 to the human or to the non-human animal. 45.A method of anti-sense therapy, which comprises administering a complexas claimed in any one of claims 1 to 30 or claim 33 to a human or to anon-human animal.
 46. A complex as claimed in any one of claims 1 to 30or claim 33 for use as a medicament or a vaccine.
 47. Use of a complexas claimed in any one of claims 1 to 30 or claim 33 for the manufactureof a medicament for the prophylaxis of a condition caused in a human ora non-human animal by a defect and/or a deficiency in a gene, or fortherapeutic or prophylactic immunisation, or for anti-sense therapy. 48.A kit that comprises (i) an integrin-binding component, (ii) apolycationic nucleic acid-binding component, and (iii) a lipidcomponent.
 49. A kit as claimed in claim 48, which also comprises (a) aplasmid or vector suitable for the expression of a nucleic acid, theplasmid or vector being either empty or comprising the nucleic acid, or(b) a nucleic acid.
 50. A kit as claimed in claim 48 or claim 49,wherein components (i) to (iii) are as defined in any one of claims 2 to29.
 51. Use of a lipid component as defined in any one of claims 20 to24, 27 and 28 for the manufacture of a medicament comprising (i) anucleic acid, (ii) an integrin-binding component, (iii) a polycationicnucleic acid-binding component and (iv) the lipid component.
 52. Amethod for transfecting a cell using (i) a nucleic acid, (ii) anintegrin-binding component, and (iii) a polycationic nucleicacid-binding component, characterised in that a lipid component is usedin addition to components (i) to (iii).
 53. Use as claimed in claim 51or a method as claimed in claim 52, wherein the lipid component is asdefined in any one of claims 20 to 24, 27 and
 28. 54. A method forexpressing a nucleic acid in a host cell, which comprises bringing thecell into contact with a complex as claimed in any one of claims 1 to 30or claim
 33. 55. A method for producing a protein, which comprisestransfecting a cell in vitro with a complex as claimed in any one ofclaims 1 to 30 or claim 33, wherein the nucleic acid component of thecomplex encodes the protein, allowing the cell to express the protein,and obtaining the protein.